The present invention relates to immortalized blastodermal avian cell lines as well as techniques for creating such lines and materials used in such methods. Such cell lines are desirable for creating transgenic birds and preserving germ line genomes.
Transgenic technology is a powerful tool for improving the commercial value of plants (e.g. enhancing their disease and herbicide resistance). However, attempts to improve the genetic characteristics of animals have lagged behind plants, with most of the early emphasis being on using animals as bioreactors to produce synthetic human pharmaceuticals. There has been little progress in improving the genetic quality of farm animals to date using transgenic techniques.
A particular impetus for such efforts is present in the poultry (e.g. chickens, ducks, geese, turkey, etc.) industry where there is typically high density rearing which results in increased risk of infection by diseases such as Marek's disease and avian leukosis. Commercial breeders have therefore sought to select (using natural breeding techniques) for poultry having increased disease resistance.
There had been some thought of trying to introduce foreign genes into poultry breeding lines for purposes of increasing disease resistance. For example, a germ line was infected with a mutant ALV virus that increased resistance to a particular subgroup of avian leukosis virus. See M. Federspiel et al. 65 J. Virol. 313-19 (1991). The disclosure of this publication and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
Attempts were also made to introduce selected foreign genes by cloning them into a retrovirus vector (e.g. reticuloendotheial virus or avian leukosis virus), injecting the recombinant virus into fertile eggs, allowing the virus to infect the developing embryo (e.g. primordial germ cells) thereby creating a chimeric gonad or ova, and using the resultant recombinant to try to introduce a foreign gene into the progeny. However, the poultry industry has been reluctant to commercially use this technology as the virus (in its natural state) is a pathogen, even variant replication competent virus vectors can sometimes induce tumors, and replication incompetent variants require high or repeated dosages. Also, even replication defective virus constructs can pose some risk of recombining with endogenous virus envelope and becoming replication competent. Further, these vectors are currently limited to DNA inserts of relatively small size (e.g. two kilobases or less).
There have also been attempts to inject foreign DNA into the undeveloped fertilized ovum after it is surgically removed from the hen. See M. Perry, 331 Nature (1998). However, this approach required incubating the developing embryo in a series of surrogate containers. Further, it required specialized laying flocks and extensive practice to obtain the needed surgical and technical skills.
A technically less demanding prior art approach involved the introduction of foreign DNA into the embryo after the egg had been laid. See generally J. Petitte et al., 76 Poult. Sci., 1084-92 (1997); R. Etches et al., 62 Methods Mol. Biol., 433-50 (1997); M. Naito et al., 113 J. Reprod. Fertil, 137-43 (1998); M. Nakamura et al., 67 Okajimas Folia Anat Jpn., 473-7 (1991); J. Petitte et al., 108 Development, 185-9 (1990); and U.S. Pat. Nos. 5,340,740 and 5,656,479, J. Petitte et al.
This involved the injection of genetically modified embryonic cells or primordial germ cells into the blastoderm either shortly after lay or about 48 hours later when the primordial germ cells are beginning their migration to the gonadal analagen. In this approach, blastodermal cell cultures were created which retained their ability to differentiate into functional ova or spermatozoa producing cells when incorporated into the developing embryo.
Blastodermal cell cultures of this type can be genetically modified and then injected into recipient embryos. The recipient embryos would typically have been previously modified by gamma irradiation to debilitate the endogenous primordial germ cells and give the injected cells a selection advantage in homing into the gonadal analagen. The modified cells would then mature and produce spermatozoa or ova capable of transmitting the transgene to at least the next generation (and preferably other future generations).
A key to the success of this technique is the ability to expand the embryonic blastodermal cells in culture while inhibiting their differentiation. Several groups have therefore developed culture techniques based on the addition of various cloned factors and feeder cells that allow short term (a few days to approximately two weeks) expansion of the embryonic cells while retaining their primordial germ cell phenotype. These cultured cells have been successful in producing germ line chimeras, but most attempts to express transfected DNA as transgenes using this system have been unsuccessful.
Another approach relied on the use of culture medium containing toxins.
Thus, it can be seen that there is a need for improved culture conditions for avian blastodermal cell cultures.